Novel Platform Using LC-MS and Ligand Binding Assays for - - PowerPoint PPT Presentation

Novel Platform Using LC-MS and Ligand Binding Assays for Characterization and PK analysis of ADCs

Jasja Wolthoorn, TNO Triskelion BV EBF Barcelona, 22nd November 2013

THE DEVELOPMENT OF LARGE MOLECULES

Recombinant proteins Antibody - Drug Conjugates Therapeutic Antibodies Novel scaffolds Biosimilars/ Biobetters

THE CYTOTOXIC DRUG Cytotoxic drugs

very efficient but unspecific – Off-target effects

Drug Mechanism

Doxorubicin derivatives Inhibit DNA religation, leading to DNA double-strand breaks Maytansinoids; Auristatins Prevent tubulin polymerization Calicheamicins Cause double-strand DNA breaks CC-1065 Induces adenine alkylation Duocarmycins Break down adenine-specific molecules in the DNA structure Anthracyclines Inhibit DNA and RNA synthesis by intercalating between base pairs, preventing replications

THE MONOCLONAL ANTIBODY Therapeutic antibodies in oncology – Very specific/targeted, But efficacy was lower than expected

THE LINKER Criteria for linker in ADC:

• Stable in blood
• Able to release effector drug at target

EXAMPLES CURRENT LINKERS

Linker Release Mechanism Hydrazone degradation in acidic compartments within the cytoplasm Peptide enzymatic hydrolyzed by lysosomal proteases such as cathepsin B Disulfide Cleavable through disulfide exchange with an intracellular thiol, such as glutathione Thioether Nonreducible and designed for intracellular proteolytic degradation Hydrophilic improve activity against multidrug resistant cells and carry a higher maytansinoid load DNA alkylator DNA-specific binding that increases reactivity, deactivates the cytotoxic, and reactivates only after the cytotoxic is cleaved

Release mechanism linker – drug will impact in vivo behavior

ANTIBODY-DRUG CONJUGATE

Antibody

Cytotoxic drug Linker

Combining the specificity of a mAb with the efficacy of a cytotoxic drug

BASIC MECHANISM ADCS

http://www.seattlegenetics.com/adc_technology

OFF-TARGET EFFECTS

Target dependent toxicity of normal cells (e.g. Her2)

Target independent toxicity via Mannose binding receptor (glycosylation mAb)

Premature release of drug

BIOANALYSIS

Why is bioanalysis assessment of ADCs challenging?

BIOANALYSIS

• Heterogeneity – more than one linker attached

to mAb – varying drug-to-antibody ratio (DAR)

• Biotransformation – dynamic in vivo behavior

WHAT TO EXPECT IN VIVO?

ADC mAb mAb + linker free drug linker metabolites drug + linker

WHAT TO EXPECT IN VIVO?

Which of these species is responsible for safety and efficacy?

BIOANALYSIS Typical approach to measure ADCs in matrix is multi- disciplinary:

Linker Definition Assay type Total antibody DAR ≥ 0 LBA Conjugated antibody DAR ≥ 1 LBA Antibody- conjugated drug Total small molecule drug conjugated to antibody Affinity LC-MS/MS, LBA Free drug Unconjugated drug in circulation LC-MS/MS Total drug Conjugated & unconjugated drug LC-MS/MS

CONJUGATED AB DAR ≥ 1

Labelled anti-Fc antibody Coated Anti-toxin antibody ADC

TOTAL MAB DAR ≥ 0

Labelled anti-mAb Coated Anti-Fc antibody ADC

IMPROVING SPECIFICITY ASSA Y

Use of anti-idiotypic reagents Anti-idiotypic = structure directed against the idiotopes Idiotopes: : unique set of antigen determinants anti-idiotypic antibody anti-idiotypic Fab fragment

TOTAL MAB WITH ANTI-IDS

Labelled anti-mAb Anti-ID antibody ADC

TOTAL MAB WITH ANTI-IDS

Anti-ID Fab ADC Labeled Anti-ID Fab

MULTI-DISCIPLINARY APPROACH

Time Free Drug Molar Concentration

Total antibody assay LBA Conjugated antibody assay LBA

Total antibody - Conjugated antibody = deconjugated Ab Information gained: average ADC in vivo fate/ no DAR distribution

LC-MS

BIOANALYSIS

Problem ligand binding assay:

• Reference standard may not be appropriate for quantification
• f analytes in vivo
• Possible solution: calibrator with most anticipated DAR form

(engineered homogenous ADC)

• High payload (drug) may interfere with assay (steric hindrance)

BIOANALYSIS

Problem LC-MS

• Measurement based on fixed mass (e.g. intact drug alone)
• Only a fraction of the cytotoxic drug may be measured

due to interaction with matrix components

• r linker partially attached to drug – most dominant form of

released drug may not be measured

• Free drug concentration normally very low (~ 1%)

NOVEL BIOANALYSIS APPROACH

Combine LC-MS with immuno pull down/ affinity capture

• Opportunity to measure ADCs and

their biotransformation in vivo over time

GENERIC CAPTURE & LC-MS/MS Generic: capture antibody (unspecific with Protein A/G), cleave drug/digest and measure on peptide level

ADC and Endogenous IgG Capture by Protein A (resin) LC-MS/MS detection Linker cleavage/ ADC digestions step

Still no DAR distribution!

GOAL – MEASURE DAR DISTRIBUTION

25

Time

21 (days) 7 1

STEP 1: SPECIFIC AFFINITY CAPTURE

Beads coated with target

+ serum ADC Magnetic separation & removal of beads Injection intact ADC into LC-MS

IMPROVING SPECIFICITY

Beads coated with anti-idiotypic antibodies

STEP 2: LC-MS OF INTACT ADC

deconvolution

representativeTIC

• f ADC (DAR 0-2)

in rat plasma

WHICH APPROACH AT WHICH STEP?

Average DAR DAR distribution LBA with Generic reagents Generic capture/ digest & LC-MS Affinity Capture (nano) LC-MS LBA with Anti- idiotypic reagents LC-MS Free drug +

R&D Preclinics Clinics

THANK YOU FOR YOUR ATTENTION